Production of L-phenylacetylcarbinol (L-PAC) by encapsulatedSaccharomyces cerevisiae cells

In the present study, benzaldehyde was converted by both the free cellsSaccharomyces cerevisiae (ATCC 834) and those immobilized in the calcium alginate liquid-core capsule intoL-PAC during anaerobic fermentation in a medium containing benzaldehyde. In a free cells survey, skipping aerobic adaptation before anaerobic fermentation caused all of benzaldehyde to be converted by 220 g (wet weight) of cells in 100 mL of the medium even at a higher concentration of 8 g/L benzaldehyde. The yield of L-PAC based on the moles of converted benzaldehyde increased as the amount of benzaldehyde dose was increased. The encapsulation protected cells effectively from the toxicity of benzaldehyde. Even a small quantity, 1.1 g (dry weight), of encapsulated cells in 100 mL of the medium containing 0.6% benzaldehyde converted more than 95% of the benzaldehyde, and the corresponding yield of L-PAC was about 40%. The production of L-PAC by the encapsulated cells depended on the pH of the medium. The conversion of benzaldehyde decreased slightly, but yield of L-PAC increased as the pH of the broth solution was fixed at a lower value. Biotransformation in a small side reactor of the batch system caused higher yield of L-PAC than that in the batch reactor containing the same quantity of encapsulated cells during the first 4 hours of fermentation.     

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.     

酵母细胞连续发酵过程振荡现象的研究进展

振荡现象在连续发酵过程中普遍存在,本文综述了酿酒酵母连续发酵中不同类型振荡现象的研究进展。一类以乙醇连续发酵过程中参数振荡为代表的特殊振荡现象,普遍存在于工业发酵过程,对发酵系统的稳定运行造成不利影响。本文论述了这类振荡现象产生的机理、调控策略的研究进展及其潜在的应用价值。     

Effects of moderate pressure on premeability and viability of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

With CO₂ and N₂ as the pressure media, the effects of the moderate pressure (0.1–1.0MPa) and the holding time on the conductivities of the cell suspension of Saccharomyces cerevisiae CICC1447 and Saccharomyces cerevisiae CICC1339, as well as the absorbances of the supernatant (after centrifuged) at 280 nm (A₂₈₀) and 260 nm (A₂₆₀) were determined. The membrane permeability of Saccharomyces cerevisiae CICC1447 increased significantly and the cell leakage was aggravated with the pressure increase. For Saccharomyces cerevisiae CICC1339, the conductivity of the cell suspension, A₂₈₀ and A₂₆₀ of the supernatant fluctuated with the pressure increase; as a whole, they increased with pressure. Different from high pressure, a moderate pressure not only remarkably improved the permeability of the yeast cell membrane, but also kept yeast cell viability; moreover, the integrity of the yeast cell membrane could be maintained. The first author and the fifth author contributed to the work equally.     

Increased production of S-adenosyl-L-methionine using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> sake K6

S. cerevisiae sake K6 was the firstly isolated industrial strain to overproduce S-adenosyl-L-methionine (SAM). Although the strain has advantages over other strains, such as GRAS (generally recognized as safe) property, the S. cerevisiae K6 has not been further developed with DNA recombinant technology due to the lack of a proper genetic marker. In this study, UV mutagenesis was conducted with S. cerevisiae sake K6. With the method, a mutant of sake yeast with leucine auxotroph, K6-1, was isolated. The mutant showed comparable growth rate and SAM productivity with its wild type. Using the auxotroph as a genetic marker, a SAM synthase in S. cerevisiae, SAM2, was overexpressed in the mutant strain. This recombinant DNA technology successfully increased SAM productivity in sake yeast. This article is dedicated to Professor Chul Soo Lee in commemoration of his retirement from Department of Chemical and Biological Engineering of Korea University.     

Different Reactive Oxygen Species Lead to Distinct Changes of Cellular Metal Ions in the Eukaryotic Model Organism Saccharomyces cerevisiae

Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al(3+)) level rose up to 50-fold after the diamide treatment. Cellular potassium (K(+)) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al(3+) accumulation was further validated by the enhanced Al(3+) uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al(3+) uptake, suggesting Al(3+)-specific transporters could be involved in Al(3+) uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.     

Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.     

Production of linolenic acid in yeast cells expressing an omega-3 desaturase from tung (<Emphasis Type="Italic">Aleurites fordii</Emphasis>)

Tung oil is an industrial drying oil containing ca. 90% PUFA. We previously reported on enzymes required for the synthesis of linoleic (6% of FA) and eleostearic (80%) acids and here describe the cloning and functional analysis of an omega-3 FA desaturase (FAD3) required for the synthesis of linolenic acid (1%). The tung FAD3 cDNA was identified by screening a tung seed cDNA library using the polymerase chain reaction and degenerate primers encoding conserved regions of the FAD3 enzyme family. Expression of this cDNA in yeast cells, cultured in the presence of linoleic acid, resulted in the synthesis and accumulation of linolenic acid, which accounted for up to 18% w/w of total cellular FA. Tung FAD3 activity was significantly affected by cultivation temperature, with the greatest amount of linolenic acid accumulating in yeast cells grown at 15°C. The amount of linolenic acid synthesized in yeast cells by tung FAD3 is ca. 10-fold higher than that observed by expression of a rapeseed (Brassica napus) FAD3 in yeast, suggesting that tung FAD3 might be useful for biotechnological production of omega-3 FA in transgenic organisms.     

<Emphasis Type="Italic">Ricinus communis</Emphasis> Contains an Acyl-CoA Synthetase that Preferentially Activates Ricinoleate to Its CoA Thioester

As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain complete coding regions corresponding to 694 and 690 amino acids, respectively. The third cDNA, RcACS 1, encodes a truncated gene sequence. The RcACS 2 and RcACS 4 share 77% identity at the amino acid sequence level. Complementation tests showed that both RcACS 2 and RcACS 4 successfully restored growth of a yeast mutant strain (YB525) deficient in ACS. Lysates from yeast cells expressing RcACS 2 and 4 were enzymatically active when using ¹⁴C-labeled oleic acid as a substrate. A cell fractionation study indicates that RcACS 2 and 4 are mainly associated with membranes. Substrate specificity assays indicate that the RcACS 2 preferentially activates ricinoleate, while the RcACS 4 has a preference for nonhydroxy fatty acids.     

氟虫腈的色谱分析方法

对氟虫腈的色谱分析方法进行了系统研究。高效液相色谱法选用紫外检测器和C18反相柱，采用外标法对市售氟虫腈产品进行定性定量分析，方法的相对标准偏差为0．42％，添加回收率为100．5％～104．3％。气相色谱法采用OV-17色谱柱和火焰离子化检测器，选用邻苯二甲酸二丁酯为内标物对市售氟虫腈样品进行定性定量分析，方法的相对标准偏差为1．60％，添加回收率为98．1％～100．8％。     

Effect of CLA on the cellular lipids of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast Saccharomyces cerevisiae was cultivated in the presence of free CLA that was either a pure trans-10, cis-12 isomer, a pure cis-9, trans-11 isomer, or a 1∶1 mixture of the two, and the influence of these supplementations on the content and FA composition of the lipids in the yeast was determined. Neither the pure isomers nor their 1∶1 mixture influenced the growth of the yeast, but the trans-10, cis-12 isomer reduced the amount of cellular lipids by 40%. The reduction in total cellular lipids by the trans-10, cis-12 CLA was due to a reduction in TAG. Both of the isomers were incorporated into the yeast lipids, reaching a proportion of about 33% in TAG. With the incorporation of CLA, the yeast reduced the amount and desaturation of endogenously synthesized FA. These clear and pronounced isomer-specific effects of CLA on the yeast suggest that yeast might be a useful model to obtain a more comprehensive view of the mechanisms of the action of CLA on lipid metabolism.     

Toxicity of allelopathic monoterpene suspensions on yeast dependence on droplet size

The toxic effects of the allelopathic nonsubstituted monoterpenes -pinene and limonene on yeast,Saccharomyces cerevisiae, were proportional to the size of the monoterpene droplets in suspension. Both the toxic effects and the size of the droplets in suspension were decreased by adding different solvents with the monoterpene as follows: dimethylsulfoxide – dimethylformamide ethanol > dioxane. Oxygen consumption was inhibited about 80% by 1 mM -pinene added in dimethylsulfoxide but less than 10% when -pinene was added in dioxane. Parallel decreases in droplet size and toxic effects of either monoterpene were also induced by hydrating the monoterpene-dimethylformamide or monoterpene-dimethylsulfoxide before addition to yeast. Molecular aggregation may be a mechanism to potentiate the allelopathic properties of monoterpenes when these associate with diverse soil components.     

Chromatographic analysis of species specific odor profiles inMastomys natalensis andM. coucha (Rodentia: Muridae)

Whole-body volatiles from males of the cryptic multimammate mouse speciesMastomys natalensis andM. coucha were analyzed by dynamic solvent effect sampling and capillary gas chromatography. One compound, 3-nonene-2-one, was always present, sometimes as the major component, in volatiles fromM. coucha and absent, or present only at low levels, in volatiles fromM. natalensis. The mean ±SD of the 3-nonen-2-one peak area forM. coucha was 8599 ±9630 and forM. natalensis 148 ±486. Chromatographic analysis was more reliable in identifying a male's species than were a female's in a two-choice olfactorium.     

Photoaffinity Labeling and Mutational Analysis of 24-C-Sterol Methyltransferase Defines the AdoMet Binding Site

Photolabeling and site-directed mutagenesis were performed on recombinant Saccharomyces cerevisiae 24-C-sterol methyltransferase (SMT) to elucidate the location and role of active site residues involved in AdoMet binding and catalysis. Bioinformatic analysis of the SMT revealed a ten amino acid segment, conserved between L124 and P133, associated with the Rossmann-like fold belonging to AdoMet-dependent methyltransferases. Irradiation of the SMT in the presence of [methyl-(3)H(3)]AdoMet directly photolabeled the protein. The specificity of photolabeling was demonstrated by inactivation experiments with structural analogs of AdoMet, including sinefungin. Trypsin digestion of the [methyl-(3)H(3)]AdoMet photolabeled Erg6p afforded a single radioactive band in SDS-PAGE gel of 4 kDa. HPLC purification of this material generated a single radioactive fraction. The corresponding (3)H-AdoMet-peptide adduct was subjected to Edman sequencing and the first fifteen residues gave a sequence Gly(120)-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala(134) that contained the predicted AdoMet binding site. Amino acid residues in the tryptic digest fragment considered to bind covalently with the AdoMet at Asp125, Cys128, Pro133 and Tyr153 were replaced with leucine and analyzed kinetically and by photolabeling inactivation experiments. The results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet.     

Alternating current electrophoretic deposition of Saccharomyces cerevisiae cells and the viability of the deposited biofilm in ethanol production

In this work, we have explored the possibility of using asymmetrical alternating current electrophoretic deposition (AC-EPD) process, which we have previously reported for enzyme deposition, to immobilize Saccharomyces cerevisiae (S. cerevisiae) cells onto stainless steel substrates. The deposition of S. cerevisiae cells at 30 Hz and 200 V_p-p permits the formation of 89 ± 16 μm thick cell layers in 30 min. The mass of the deposited cells is shown to increase quasi-linearly with the deposition time and the applied amplitude. In order to increase the mechanical stability of the immobilized cells, a thin layer of polyurethane was applied after the AC-EPD of S. cerevisiae cells. The viability of the immobilized cells was tested in the production of ethanol. The results showed that the fermentation process with the immobilized S. cerevisiae cells is more efficient than the fermentation carried out with similarly treated free cells.     

Chemical stimuli in host-habitat location byLeptopilina heterotoma (Thomson) (Hymenoptera: Eucoilidae), a parasite ofDrosophila

Chemical stimuli play an important role in the process of searching for a host habitat by parasitic wasps. Volatile compounds originating from host habitats and/or hosts are the cues that enable such a location.Leptopilina heterotoma, a larval parasite ofDrosophila, is attracted to the food of its host, baker's yeast. Analysis of the fermentation products of baker's yeast, using a mass spectrometer, and olfactometer studies indicate that three fermentation products of this yeast, the main component of the host habitat in our laboratory, attractL. heterotoma: ethanol (5%), ethyl acetate (10^–2, 10^–3%), and acetaldehyde (1%). A combination of these three compounds, however, cannot compete with baker's yeast in attracting the parasites. Thus other factors, such as different compounds, concentrations, and/or combinations, also, play a role and remain to be tested.Leptopilina heterotoma does not use host-related olfactory cues in long-distance habitat location as it cannot distinguish between host habitat and host habitat with hosts.     

High-Performance Thin-Layer Chromatographic Analysis of Sugars in Snail-Conditioned Water and Mucus from Biomphalaria glabrata, Helisoma trivolvis, and Lymnaea elodes

The presence of sugars, particularly glucose, maltose, and sucrose, in the SCW of Biomphalaria glabrata, the two strains of Helisoma trivolvis, and Lymnaea elodes was variable, although only maltose was quantifiable in two samples of snail conditioned waters from B. glabrata. The only sugar detected in mucus was glucose, but it was below the lowest quantifiable amount in the mucus samples of B. glabrata and H. trivolvis. The mean percentage of glucose in the mucus of L. elodes was 0.0040% ± 0.00067%.     

Cloning of Flax Oleic Fatty Acid Desaturase and Its Expression in Yeast

Fatty acid desaturases dehydrogenate acyl chains, which results in the formation of a double bond. Using PCR on flax genomic DNA, we cloned a putative Δ12 fatty acid desaturase (Fad2) gene encoding a 378 amino acid protein. Heterologous expression of this protein in yeast as an N-terminal fusion to GFP showed its localization within endoplasmic reticulum. Analysis of membrane lipids revealed the production of dienoic fatty acids, decreased levels of FAD2 substrates and an increased concentration of longer fatty acids. Higher peroxidation of lipids in FAD2-containing strains is not reflected by any visible phenotype in YPD medium. However, FAD2-containing strains with deleted superoxide dismutase genes exhibited significant growth reductions under oxidative stress.     

Feasibility study of using montmorillonite for stability enhancement of l-ascorbic acid

This study tended to construct new l-ascorbic acid (LAA) composites in low toxicity and high stability for feasible application. LAA is chemically very unstable, since it is easily oxidized into biologically inactive compounds naturally. Our finding showed that introduction of montmorillonite (MMT) could significantly attenuate its toxicity and to sustain the stability of LAA with economic feasibility for practical uses. In addition, as phosphoric acid was biologically compatible, it was used for the pretreatment of MMT to obtain a promising stabilization of LAA. Toxicity assessment also showed that MMT treated with low-concentration acids should be considered as biologically safe according to our assessment. Thus, using acid treated MMT to stabilize LAA in a long-term might be technically feasible for further uses.